Each year in the United States 100,000 patients are infected with enterohemorrhagic Escherichia coli (EHEC) of the O157:H7 serotype. Infections can rapidly progress to life-threatening complications, such as hemolytic uremic syndrome, the principal cause of acute kidney failure in children, hemorrhagic colitis and central nervous system failure, frequently with lethal consequences. Our objective is to identify bacterial key pathogenicity factors underlying EHEC disease. This proposal is directed at first cataloguing/determining the lineage-wide genomic plasticity/diversity in E. coli O157:H7, and second to correlate variation in the genomic make-up to identify causable loci of Shiga toxin phage mobilization and toxin expression indicative of adverse pathogenic potential in infected patients. Stx encoded in bacteriophages and produced by the bacteria during infection plays a critical role in the evolution and pathogenesis of EHEC as direct mediators of progression to severe disease, yet it is not known how differences in the bacterial virulence genotype relate to disease manifestation. Although different typing methodologies have been developed, these assays rely on a scarce marker base, providing inadequate resolution power, and thus often fail to report the now acknowledged genetic heterogeneity in this genetically similar pathogen. This highlights the crucial need to capture the specific genetic make-up of high Stx toxin producers and to determine the genomic diversity that exists among currently circulating isolates on a lineage- and genome-wide scale. For this study we have assembled a unique resource of more than 250 E. coli O157:H7 to best represent the extant plasticity comprised of archived cultures and genomes fortified with informative clinical, environmental, and epidemiological metadata, which are available in our laboratory for in silico and biological analyses. To define the specific genetic make-up of high-level Stx toxin producer we will deploy innovative whole genome sequence typing strategy utilizing a custom polymorphism discovery pipeline to enrich the database of mutational markers and determine Stx phage virulence profiles of isolates derived from patients, contaminated produce and zoonotic reservoir. We have further developed an assay to characterize the pathogenic potential of isolates as determined by their phenotypes in Stx phage mobilization and toxin expression. Genome-wide association of the individual bacterial genotypes and Stx phenotypes are foundational to unravel causable bacterial variant loci of Stx mediated EHEC pathogenesis. In conclusion, we envision that our findings will have immediate impact on the decrease of human morbidity rates. Suppression of toxin production is an attractive target for therapeutics modifying disease outcome. The enriched catalogue of genomic variants will serve as a critical resource to precisely partition this globally emerging pathogen in to subgroups genotypic and phenotypic traits and enables us to test whether particular genotypes reflect the presence of highly virulent clones among circulating pathotypes for improved diagnostic risk assessment and biosurveillance.